CTSB was overexpressed in Nthy-ori-3-1 and 8505C cells cultured in 3 mL media of 6-well plates at a density of 4 × 105 cells per well. After 16 h, 2 μg of pCS4-3xMyc-CTSB plasmid was transfected using Lipofectamine2000 (Invitrogen, CA) according to manufacturer’s guidelines. CTSB expression was knocked down using siRNA in SNU790 and TPC-1 cells. Both siCTSB (sc-29238) and a scrambled control siRNA (sc-37007) were obtained from Santa Cruz Biotechnology Inc (Santa Cruz, CA, USA). Cells were seeded in 6-well plates at a density of 4 × 105 cells per well. After 16 h, transfection was performed using the TransIT-X2® Transfection kit (Mirus, MIR6000, Madison, WI, USA), according to manufacturer’s instructions. After 48 h, transfected cells of plasmid or siRNA were harvested.
Transit x2 transfection kit
Manufactured by Mirus Bio
Sourced in United States
The TransIT-X2® Transfection kit is a laboratory reagent used for the delivery of nucleic acids, such as DNA or RNA, into cells. It facilitates the efficient transfer of genetic material into a variety of cell types for the purpose of gene expression, gene silencing, or other research applications.
Automatically generated - may contain errors
Lab products found in correlation
Scrambled control sirna sc 37007, by Santa Cruz Biotechnology (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Non targeting sirna, by Thermo Fisher Scientific (1 mentions)
Ambion silencer sirna, by Thermo Fisher Scientific (1 mentions)
Transit mrna transfection kit, by Mirus Bio (1 mentions)
1.2 na objective, by Olympus (1 mentions)
3 protocols using transit x2 transfection kit
1
Overexpression and Knockdown of CTSB in Thyroid Cell Lines
2
Silencing Host Factors to Modulate Viral Production
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For gene silencing, three unique Ambion Silencer siRNAs (Thermo Fisher Scientific) targeting 13 host factors identified by AP-MS were pooled and transfected into THP-1 macrophages at a final concentration of 25 nM. To simultaneously knock down G3BP1 and G3BP2 as positive control, two unique Ambion Silencer siRNAs respectively targeting G3BP1 and G3BP2 were pooled (25 nM) and transfected into THP-1 macrophage. The same amount of non-targeting siRNA (Thermo Fisher Scientific) was transfected into THP-1 macrophages as negative control. siRNA transfections were performed with TransIT-X2 Transfection Kit (Mirus Bio) following manufacturer’s instructions. Downstream assays were conducted 48 h after transfection.
To observe viral production in transfected macrophages, 500 ng of viral genomic RNA was transfected per well in 12-well plates through the TransIT®-mRNA Transfection Kit (Mirus Bio) following manufacturer’s instructions.
To observe viral production in transfected macrophages, 500 ng of viral genomic RNA was transfected per well in 12-well plates through the TransIT®-mRNA Transfection Kit (Mirus Bio) following manufacturer’s instructions.
3
Optical Trap Tether Pulling Experiments
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Tether pulling experiments were performed on a home-built optical trap, following principles described elsewhere 65 (link), 96 . Briefly, 4 µm anti-Digoxigenin coated polystyrene beads (Spherotech) were trapped with a 1064 nm, Ytterbium laser (IPG Photonics) focused through a 60x 1.2 NA objective (Olympus). Forces on the beads were measured by the deflection of backscattered trapping laser light onto a lateral effect position sensor (Thorlabs) and calibrated using the viscous drag method 97 (link). To measure tether radii (R), cell lines were transiently transfected with a membrane-targeted fluorescent protein (glycosylphosphatidylinositol-anchored eGFP, Addgene #32601) using a TransIT-X2 transfection kit (Mirus). Tether radius was obtained by comparing tether fluorescence to fluorescence counts from a known area of the parent cell membrane, as described 48 (link). Tether force (f) and fluorescence measurements were performed simultaneously. Membrane tension was calculated using the following equation:
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